Organ procurement currently poses one of the major problems in organ transplantation, as the number of patients requiring transplants far exceeds the number of organs available. Xenotransplantation may provide a solution to this problem. Phylogenetically, non-human primates are the most closely related species to humans and might therefore represent the first choice as donors. In 1969, Reetsma et al. achieved the first successful kidney human xenograft from a chimpanzee (Reetsma, K. et al., 1964, Ann. Surg. 160:384). However, the potential utilization of primate donors is limited by insufficient numbers, legal and ethical considerations, and the potential for transmitting dangerous viral diseases. Swine represent one of the few large animal species in which breeding characteristics make genetic experiments possible, making it possible to develop MHC homozygous lines of miniature swine. Miniature swine can be maintained at maximum adult weights of 200 to 300 lbs and are anatomically and physiologically close to humans. Therefore the organs of miniature swine seem appropriate for use as xenografts for human beings of all ages.
Tolerance to self major histocompatibility (MHC) antigens occurs during T cell maturation in the thymus (McDuffie et al., J. Immunol. 141:1840, 1988). Exposure of the immune system to MHC antigens during ontogeny can cause the immune system to lose reactivity to those antigens, thus leaving the animal specifically tolerant into adult life (Billingham et al., 1953, Nature 172:603). Transplantation immunologists have sought means of inducing tolerance in adult animals by production of lymphohematopoietic chimeras. The induction of tolerance across MHC barriers in adult mice by whole body irradiation (WBI) and bone marrow transplantation (BMT) has been studied extensively in murine models (Hayfield et al., 1983, Transplan. 36:183; Mayumi et al., 1989, J. Exp. Med. 169:213; Sykes et al., 1988, Immunol. Today 9:23).
The use of MHC mismatched BMT as a means of inducing tolerance to organ grafts can be accompanied by several major disadvantages: the preparative regimen involves lethal irradiation, with its inherent risks and toxicities; clinical applicability is limited by the fact that most potential recipients do not have an appropriate MHC-matched donor, and BMT across MHC barriers causes severe graft-vs-host-disease (GVHD). Removing the T lymphocytes in allogeneic bone marrow inocula (Rodt et al., 1971, Eur. J. Immunol. 4:25) to prevent GVHD is associated with increased rates of engraftment failure (Martin et al., 1988, Bone Marrow Transplant 3:445; O'Reilly et al., 1985, Transplant. Proc. 17:455; Soderling et al., 1985, J. Immunol., 135:941). While these drawbacks are generally considered acceptable for the treatment of otherwise lethal malignant diseases, they would severely limit the application of MHC mismatched BMT as a preparative regimen for organ transplantation, in which non-specific immunosuppressive agents, while not without major complications, are effective.
Use of a relatively non-toxic, non-myeloablative preparative regimen for bone marrow engraftment and specific transplantation tolerance has been applied to the concordant rat to mouse species combination (Sharabi, Y. et al., 1990, J. Exp. Med. 172:195-202). The treatment involved administration of monoclonal antibodies to eliminate mature T cell subsets (CD4 and CD8) as well as NK cells (NK1.1). These monoclonal antibodies permitted engraftment of xenogeneic bone marrow after only a sub-lethal (300 rads) dose of WBI and a local dose of 700 rads thymic irradiation. The resulting lymphoid reconstitution was superior to that of previously mixed xenogeneic chimeras prepared by lethal irradiation and reconstitution with mixtures of T cell-depleted syngeneic and xenogeneic bone marrow (Sharabi, Y., et al., 1990, J. Exp. Med. 172:195-202; Ildstad, et al., 1984, Nature 307:168-170) as recipients did not suffer toxic effects from the preparative regimen. In addition, attempts have been made to lengthen the survival of skin allografts in primates and man by intravenously administering a polyclonal preparation of horse anti-human antithymocyte globulin (ATG). The ATG was injected simultaneously with and on days immediately following grafting (Cosimi, A. B. et al., 1970, Surgery. 68:54-61).
It has been recognized that the use of swine organs for xenogeneic transplantation to humans is facilitated by inducing tolerance (i.e., reducing the severity of and/or eliminating any immunological response to the transplant) to swine tissue using swine bone marrow. The swine bone marrow cells (BMC) can be transplanted to the recipient's marrow and engraft there. Engraftment, as used herein, refers to implantation or transplantation of porcine BMCs into a xenogeneic recipient or host such that the porcine BMCs proliferate, differentiate and function as bone marrow in the recipient. The porcine bone marrow can be introduced before transplantation of the swine organ, contemporaneously with the organ transplantation, or both. In this context, contemporaneously or substantially contemporaneously contemplates introduction during the same operative procedure or as part of preoperative preparation.
In accordance with the present invention, it has been recognized by the inventors that it would be highly desirable to promote the engraftment of the porcine bone marrow and that cytokines which have an effect on marrow engraftment are highly species specific in their effect. In accordance with the invention, the inventors recognized the deficiency that porcine cytokines effective to promote porcine bone marrow engraftment had not been identified, isolated, characterized or produced, such as by recombinant techniques and that such was highly desirable for use in the above and other applications.
Accordingly, other principal aspects of the invention are porcine cytokines that preferentially enhance the proliferation and engraftment of porcine bone marrow cells in the presence of bone marrow cells of other species, DNA sequences therefor and DNA molecules for expression of these porcine cytokines. More particularly, the invention provides porcine chimeric enhancement factors ("CHEFs") that are porcine analogs of interleukin-3 (hereinafter "CHEF-1"), granulocyle-macrophage colony stimulating factor (hereinafter "CHEF-2") and stem cell factor (hereinafter "CHEF-3") as well as combinations of these novel porcine cytokines with each other and with other porcine cytokines, such as porcine leukemia inhibitory factor (hereinafter "porcine LIF"). The porcine cytokines of the invention are contemplated to encompass the protein whether purified from native origin, expressed by recombinant methodologies or chemically synthesized.
As will be explained in more detail below, the porcine bone marrow that is preferentially stimulated by the porcine cytokines in the recipient prepares the recipient for the tissue or organ transplantation by inducing tolerance at both the B-cell and T-cell levels. Preferably, the bone marrow cells include immature cells (e.g., undifferentiated hematopoietic stem cells; these cells can be separated out of the bone marrow prior to administration), or a complex bone marrow sample including such cells can be used.
Preferred embodiments include those in which: swine of the same immunological profile are the donor of both the tissue or organ to be transplanted and the bone marrow; the recipient mammal is a primate, preferably a human; and the swine is a partially or completely inbred strain, e.g., a miniature swine. In a preferred embodiment of the method of use, the recipient is irradiated with low dose radiation prior to introducing the bone marrow, preferably with radiation of more than 100 rads and less than 400 rads.